by 1 to 2% per year, and commercially available vaccines often do not elicit protection from one year to the next, necessitating
frequent formulation changes. This represents a major challenge to the development of a cross-protective vaccine that can
pro-tect against circulating viral antigenic diversity. We have constructed a recombinant modified vaccinia virus Ankara (MVA) that
expresses an H5N1 mosaic hemagglutinin (H5M) (MVA-H5M). This mosaic was generated
in silico
using 2,145 field-sourced
H5N1 isolates. A single dose of MVA-H5M provided 100% protection in mice against clade 0, 1, and 2 avian influenza viruses and
also protected against seasonal H1N1 virus (A/Puerto Rico/8/34). It also provided short-term (10 days) and long-term (6
months) protection postvaccination. Both neutralizing antibodies and antigen-specific CD4
ⴙand CD8
ⴙT cells were still
de-tected at 5 months postvaccination, suggesting that MVA-H5M provides long-lasting immunity.
IMPORTANCE
Influenza viruses infect a billion people and cause up to 500,000 deaths every year. A major problem in combating influenza is
the lack of broadly effective vaccines. One solution from the field of human immunodeficiency virus vaccinology involves a novel
in silico
mosaic approach that has been shown to provide broad and robust protection against highly variable viruses. Unlike a
consensus algorithm which picks the most frequent residue at each position, the mosaic method chooses the most frequent
T-cell epitopes and combines them to form a synthetic antigen. These studies demonstrated that a mosaic influenza virus H5
hem-agglutinin expressed by a viral vector can elicit full protection against diverse H5N1 challenges as well as induce broader
immu-nity than a wild-type hemagglutinin.
I
nfluenza viruses are significant health concerns for animals and
humans. The World Health Organization estimates that every year
influenza viruses infect up to 1 billion people, with 3 million to 5
million cases of severe disease and 300,000 to 500,000 deaths
occur-ring annually (
1
). Highly pathogenic avian influenza (HPAI) H5N1
viruses have spread as far as Eurasia and Africa since their first
emer-gence in 1996. These viruses infect a range of domestic and wild avian
species as well as mammals (
2
) and pose a pandemic threat (
3
).
Cur-rent prevention and treatment strategies for H5N1 virus either are
antiviral or vaccine based or involve nonpharmaceutical measures,
such as patient isolation or hand sanitation (
4
,
5
). However, these
approaches have flaws (
5–7
), such that a broadly effective strategy for
H5N1 control remains elusive.
A powerful tool for preventing future H5N1 pandemics would
be a universal H5N1 vaccine. The generation of inactivated
vac-cines (INVs) has been optimized for seasonal flu but presents
several challenges for H5N1 viruses, including the following: (i)
the continual evolution of the viruses makes predicting a vaccine
strain difficult, (ii) egg propagation of vaccine stock is hindered
due to the high lethality of H5N1 viruses to eggs and the poultry
that provide them, and (iii) the 6- to 9-month time period
re-quired to produce INVs may be too long to protect large
popula-tions during a pandemic. In addition, initial studies in mice and
ferrets and phase 1 human clinical trials have demonstrated that
INVs and other split-virion vaccines may require higher doses of
antigen than traditional INVs, with more than one administration
being needed to provide protective immunity (
8
,
9
). Live vaccines
elicit both humoral and cellular immune responses. However,
they are not recommended for use in infants or in elderly or
immu-nocompromised individuals because they can cause pathogenic
reac-tions (
10
,
11
). Moreover, the viruses in live vaccines can revert to
wild-type (wt) viruses, potentially leading to vaccine failure and
dis-ease outbreaks (
12
). Thus, the development of new vaccine vectors
and novel approaches to antigen expression are urgently needed to
generate an effective H5N1 vaccine with broad cross-protective
effi-cacy. The modified vaccinia virus Ankara (MVA) vector offers several
advantages, such as (i) safety, (ii) stability, (ii) rapid induction of
humoral and cellular responses, and (iv) the ability to be given by
multiple routes of inoculation (
13–15
). In addition, we and others
have previously demonstrated the suitability of using MVA as a
vac-cine vector against H5N1 viruses (
14
,
16
).
Multiple approaches to the development of a universal
influ-enza vaccine that could be applied to H5N1 viruses have been
studied. One approach is to use conserved sequences, such as the
stalk region of hemagglutinin (HA) (
17–19
) or the internal
nu-cleoprotein (NP) or M1 protein (
20
,
21
). Another approach
in-volves consensus sequences that combine many H5N1
hemagglu-Received29 May 2014Accepted26 August 2014
Published ahead of print10 September 2014
Editor:D. S. Lyles
Address correspondence to Jorge E. Osorio, osorio@svm.vetmed.wisc.edu.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JVI.01532-14
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tinin sequences into a single gene. Of these approaches, only the
approach with consensus sequences has been shown to provide
partial protection against a diverse panel of H5N1 isolates (
22
).
Recently, a novel
in silico
mosaic approach has been shown to
provide broad and robust protection against highly variable viruses
(
23
). The method uses a genetic algorithm to generate, select, and
recombine potential CD8
⫹T cell epitopes into mosaic proteins that
can provide greater coverage of global viral variants than any single
wild-type protein. This approach has been able to achieve between
74% and 87% coverage of HIV-1 Gag sequences, whereas a single
natural Gag protein achieved only 37% to 67% coverage (
23–25
).
Results in studies with rhesus monkeys showed that mosaic
se-quences increased both the breadth and the depth of cellular immune
responses compared to the breadth and the depth of the responses
achieved with consensus and natural sequences (
23
).
In this study, we used an MVA vector to express a mosaic
H5N1 HA gene. In mice, a single dose of an MVA construct that
expresses an H5N1 mosaic hemagglutinin (H5M) (MVA-H5M)
provided sterilizing immunity (no virus was detectable in lung
tissues postchallenge) against H5N1 HPAI clade 0, 1, and 2
vi-ruses. Furthermore, MVA-H5M provided full protection at as
early as 10 days postexposure and for as long as 6 months
postvac-cination. Both neutralizing antibodies and antigen-specific CD4
⫹and CD8
⫹T cells were detected at 5 months postvaccination. In
addition, MVA-H5M also provided cross-subtype protection
against H1N1 virus (A/Puerto Rico/8/34 [PR8]) challenge. Our
results indicate that the mosaic vaccine approach has great
poten-tial for broadening the efficacy of influenza vaccines, perhaps
in-cluding protection against multiple influenza virus subtypes.
MATERIALS AND METHODS
Cells and viruses.Chicken embryo fibroblasts (CEFs) and Madin-Darby canine kidney (MDCK) cells were obtained from Charles River
Laborato-ries, Inc. (Wilmington, WA, USA), and the American Type Culture Col-lection (ATCC; Manassas, VA, USA), respectively. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. CEFs were used for propagating MVA. Highly pathogenic avian influenza (HPAI) H5N1 virus A/Vietnam/ 1203/04 (A/VN/1203/04) was kindly provided by Yoshihiro Kawaoka (University of Wisconsin—Madison, Madison, WI, USA). Highly patho-genic avian influenza H5N1 viruses A/Hong Kong/483/97 (A/HK/483/ 97), A/Mongolia/Whooper swan/244/05 (A/MG/244/05), and A/Egypt/ 1/08 and seasonal influenza viruses, including A/Puerto Rico/8/34 (PR8; H1N1) and A/Aichi/2/1968 (H3N2), were kindly provided by Stacey Schultz-Cherry and Ghazi Kayali (St. Jude Children’s Research Hospital, Memphis, TN, USA). All viruses were propagated and titrated in MDCK cells with DMEM that contained 1% bovine serum albumin and 20 mM HEPES. Viruses were stored at⫺80°C until use. Viral titers were deter-mined and expressed as 50% tissue culture infective doses (TCID50s). All experimental studies with HPAI H5N1 viruses were conducted in a bio-safety level 3⫹(BSL3⫹) facility in compliance with the University of Wisconsin—Madison Office of Biological Safety.
Plasmid and MVA recombinant vaccine construction.A total of 3,069 HA protein sequences from H5N1 viruses available in the National Center for Biotechnology Information (NCBI) database were downloaded and screened to exclude incomplete and redundant sequences. The resulting 2,145 HA sequences were selected to generate one mosaic protein sequence, as previously described (26). Briefly, all 2,145 HA sequences were uploaded into the Mosaic Vaccine Designer tool webpage (http://www.hiv.lanl.gov/content /sequence/MOSAIC/makeVaccine.html). In the parameter options, the cocktail size was set to 1 in order to generate a single peptide that repre-sented all uploaded sequences. The rare threshold was set to 3 for optimal value, and most importantly, the epitope length parameter was set to an amino acid length of 12-mer in an attempt to match the length of natural T helper cell epitopes (27). The resulting mosaic H5N1 sequence (H5M) was back-translated and codon optimized for mice. The optimized H5M sequence was then synthesized commercially (GenScript USA Inc., Pisca-taway, NJ, USA). Transfer plasmid PI2-Red encodingDiscosomasp. red
FIG 1The mosaic H5 hemagglutinin (H5M) sequence. The mosaic H5N1 hemagglutinin (H5M) sequence was deduced from 2,145 HA sequences. The lines
above the sequence indicate known T helper cell, B cell, or cytotoxic T lymphocyte (CTL) epitopes that were found in H5M by use of the Influenza Research Database (IRD).
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[image:2.585.96.491.66.315.2](Madison, WI, USA).
Analysis of HA expressed by H5M.The hemagglutinin expressed by MVA-H5M was analyzed by Western blot analysis. CEF cells were infected at a multiplicity of infection (MOI) of 1 PFU/cell of the MVA-H5M and MVA-HA constructs and a construct consisting of MVA expressing lucif-erase (MVA-LUC). Infected cell pellets were harvested at 48 h postinfec-tion and lysed with Laemmli sample buffer (Bio-Rad, Hercules, CA, USA). Protein was fractionated via SDS-PAGE and transferred onto a nitrocellulose membrane for hemagglutinin detection by a specific an-ti-HA antibody (Ab). 3,3=,5,5=-Tetramethylbenzidine (TMB) was used to visualize the HA protein in the membranes.
Functional analysis of H5M was done by hemagglutination assay (29). CEF cells were infected at an MOI of 1 with MVA-H5M, MVA-HA, and MVA-LUC. At 48 h postinfection, cells were harvested and 2-fold dilu-tions with phosphate-buffered saline (PBS) were made in round-bottom 96-well plates. Chicken red blood cells (RBCs) were added into each well, and the plates were incubated for 30 min. Lattice formations, which are indicative of the ability of HA to agglutinate RBCs, were observed in positive wells.
Animal studies.All mouse studies were conducted at the University of Wisconsin—Madison animal facilities and were approved by the Interin-stitutional Animal Care and Use Committee (IACUC). Challenge exper-iments involving H5N1 viruses were conducted at animal BSL3⫹ (ABSL3⫹) facilities. Challenge studies with seasonal influenza, PR8 (H1N1), and A/Aichi/2/1968 (H3N2) viruses were conducted under BSL2 conditions to facilitate animal monitoring.
Vaccine efficacies.In the first study, groups of 5-week-old BALB/c mice were vaccinated with 1⫻107PFU of either recombinant MVA-H5M or MVA-LUC via the intradermal (i.d.) route (8 mice per group). Intra-dermal inoculations were done by injecting 50l of PBS containing virus into footpads. At 4 weeks after vaccination, blood samples were collected for serological analysis. At 5 weeks postvaccination, mice were challenged by intranasal (i.n.) instillation, while they were under isoflurane anesthe-sia, with 100 50% lethal doses (LD50s) of A/Vietnam/1203/04 (1⫻104 TCID50s), A/Hong Kong/483/97 (4 ⫻ 103 TCID
50s), A/Mongolia/ Whooper swan/244/05 (1⫻103TCID
50s), or A/Egypt/1/08 (3.56⫻104 TCID50s) contained in 20l of PBS. Two mice from each group were euthanized at day 5 postchallenge, and lung tissues were collected for viral titrations and histopathology. For virus isolation, lung tissues were minced in PBS using a mechanical homogenizer (MP Biochemicals, So-lon, OH, USA), and the viral titers in the homogenates were quantified by plaque assay on MDCK cells. The remaining lung tissue was fixed in 10% buffered formalin. The remaining animals in each group (6 mice per group) were observed daily for 14 days, and survival and clinical param-eters, including clinical score and body weight, were recorded. Mice show-ing at least a 20% body weight loss were humanely euthanized.
The second study evaluated the protective efficacies of the MVA-H5M vaccine against seasonal influenza virus, PR8 (H1N1), or A/Aichi/2/1968 (H3N2). Groups of 5-week-old BALB/c mice were vaccinated with MVA-H5M or MVA-LUC as described above (8 mice per group). At 4 weeks after vaccination, blood samples were collected for serological analysis. At 5 weeks postvaccination, the mice were challenged, while they were under isoflurane anesthesia, by i.n. instillation of 50l of PBS containing 100 LD50s of PR8 (6.15⫻103TCID50s) or A/Aichi/2/1968 (3.405⫻106 TCID50s). Two mice from each group were euthanized at day 3 postchal-lenge, and lung tissues were collected as described above. The remaining
animals in each group (6 mice per group) were observed daily for 14 days as described above.
In the third study, we evaluated the ability of the MVA-H5M vaccine to confer both short- and long-term protection against avian influenza virus A/Hong Kong/483/97; this strain showed the most virulence from the previous study. Groups of 5-week-old BALB/c mice were vaccinated with MVA-H5M or MVA-LUC as described above (7 mice per group). Blood samples were collected at 10 days and 6 months postvaccination for short- and long-term studies, respectively. At 10 days and 6 months post-vaccination, mice were challenged, while they were under isoflurane an-esthesia, by i.n. instillation of 100 LD50s of A/Hong Kong/483/97 (4⫻103 TCID50s). Two mice from each group were euthanized at day 5 postchal-lenge, and lung tissues were collected for viral titrations and histopathol-ogy. The remaining animals in each group (5 mice per group) were ob-served daily for 14 days, as described above.
FIG 2The MVA-H5M vaccine expresses a higher level of protein than MVA
expressing wild-type hemagglutinin (MVA-HA) and elicits broad neutralizing antibodies against avian influenza viruses. (A) Western blot analysis of CEF cell lysates infected with MVA-H5M, MVA-HA, and MVA-LUC (negative control). HA from MVA-H5M was expressed as a cleavable protein that was the same as the HA from MVA-HA. The sizes of HA clade 0 (HA0), HA clade 1 (HA1), and HA clade 2 (HA2) are 75 kDa, 50 kDa, and 25 kDa, respectively. (B) The titers of neutralizing antibodies against influenza virus A/VN/1203/04, A/MG/244/05, A/HK/483/97, A/Egypt/1/08 (EGYPT/01/08), PR8 (H1N1), or A/Aichi/2/1968 (H3N2) virus in vaccinated mice (n⫽8 mice per group) were measured at 4 weeks postvaccination. No statistically significant differences between titers against avian influenza viruses (P⬎0.05, one-way ANOVA) were found.
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[image:3.585.300.541.66.436.2]Serology.Serum antibody titers were determined by microneutraliza-tion assay. Briefly, serum was incubated at 56°C for 30 min to inactivate complement and then serially diluted 2-fold in microtiter plates. Two hundred TCID50s of virus were added to each well, and the plates were incubated at 37°C for 1 h. The virus-serum mixture was added to dupli-cate wells of MDCK cells in 96-well plates, the plates were incubated at 37°C for 72 h, and then the cells were fixed and stained with 10% (wt/vol) crystal violet in 10% (vol/vol) formalin to determine the TCID50. The titer was determined as the serum dilution resulting in the complete neutral-ization of the virus.
Histopathology and immunohistochemistry.Lung tissue samples for histological analysis were processed by the histopathology laboratory at the School of Veterinary Medicine, University of Wisconsin—Madison (Madison, WI, USA), and stained with hematoxylin and eosin. For im-munohistochemistry, tissue sections were deparaffinized and rehydrated as previously described (30). Slides were treated with antigen retrieval buffer, followed up with treatment with 3% H2O2. The slides were placed in blocking solution and incubated in goat anti-HA (1/300 dilution; Bio-defense and Emerging Infections Research Resources Repository number NR-2705) avian influenza virus polyclonal antibody for 24 h. Secondary
horseradish peroxidase-conjugated antigoat antibody at a 1/5,000 dilu-tion was added onto the slides, and the slides were incubated for 1 h. Then, the slides were stained with 0.05% 3,3=-diaminobenzidine (DAB) sub-strate to visualize the presence of avian influenza virus antigens.
T cell responses.At 6 weeks and 5 months postvaccination, MVA-H5M-vaccinated mice were euthanized (3 mice per group) and spleens were aseptically removed. Splenocytes from individual animals were sus-pended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100 IU/ml penicillin, 100g/ml streptomycin, and 0.14 mM-mercaptoethanol. Red blood cells were lysed with 1⫻BD Pharm Lyse buffer (BD Biosciences, San Jose, CA, USA). Following washing with RPMI 1640 medium, cells were resuspended in the same medium and 1⫻ 106splenocytes were surface stained with anti-mouse CD4 fluorescein isothiocyanate (RM4-5) and anti-mouse CD8a peridinin chlorophyll pro-tein (53-6.7) monoclonal antibodies (BD Biosciences, San Jose, CA, USA).
In order to study intracellular cytokine responses, 1⫻106splenocytes were plated onto a 96-well flat-bottom plate and stimulated with diverse H5N1 HA peptide pools (5g/ml) in a 200-l total volume for 16 h. Brefeldin A (BD GolgiPlug; BD Biosciences, San Jose, CA, USA) was
Egypt / 01/08
Egypt / 01 /08
Egypt / 01/08
FIG 3MVA-H5M provides broad protection against multiple clades of avian influenza virus. (A) Efficacies of a single dose of the MVA-H5M or MVA-LUC
vaccine against highly pathogenic avian influenza viruses: clade 0 influenza virus A/HK/483/97 virus, clade 1 influenza virus A/VN/1203/04, clade 2.2 influenza virus A/MG/244/05, and clade 2.2.1 influenza virus A/Egypt/1/08. Mice vaccinated with MVA-H5M showed 100% survival, and MVA-H5M prevented morbidity during the studies. (B and C) No viral titers were detectable in the lung at day 5 postinfection (B), and MVA-H5M-vaccinated mice maintained their weight throughout the studies (C). Vaccinated mice were challenged at 5 weeks postvaccination, 2 mice per group were sacrificed at day 5 postinfection, and survival data were monitored for 14 days (n⫽6 mice per group).
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[image:4.585.42.539.54.442.2]added at a final concentration of 1g/ml for the last 5 h of incubation to block protein transport. Cells were stained intracellularly for gamma in-terferon (IFN-␥) allophycocyanin (XMG1.2) and interleukin-2 (IL-2) phycoerythrin (JES6-5H4) after surface staining for CD4 and CD8a. All antibodies were from BD Biosciences (San Jose, CA, USA), except where noted. The samples were acquired on a BD FACSCalibur flow cytometer and analyzed with FlowJo (v10.0.6) software (TreeStar Inc., Ashland, OR, USA). The background cytokine level from medium-treated groups was subtracted from the cytokine level for each treated sample. The frequency of cytokine-positive T cells was presented as the percentage of gated CD4⫹ or CD8⫹T cells.
Statistical analysis.Student’sttests and one-way analysis of variance (ANOVA) were used to evaluate viral lung titers, antibody titers, and T cell responses between groups. Survival analyses were performed to assess vaccine effectiveness against challenge viruses. Probability values of⬍0.05 were considered significant. GraphPad Prism (v6) software (La Jolla, CA, USA) was used for all statistical analyses.
RESULTS
Construction of pox-based H5N1 mosaic hemagglutinin
vac-cine.
A mosaic vaccine that targets the hemagglutinin protein of
influenza H5N1 virus was constructed (
Fig. 1
). The HA mosaic
was generated using an input of 2,145 HA sequences from H5N1
influenza viruses available in GenBank. To maximize T helper cell
epitope coverage, the
in silico
algorithm was set to an amino acid
length of a 12-mer (
27
). The mosaic H5 (H5M) sequence was
back-translated into DNA and cloned into modified vaccinia virus
Ankara (MVA) to generate MVA-H5M. Recombinant H5M was
expressed as cleavable HA that resembled wild-type (wt) HA from
avian influenza virus A/VN/1203/04, as shown in
Fig. 2A
.
Inter-estingly, the level of protein expression from MVA-H5M-infected
cell pellets was higher than that from MVA expressing wild-type
hemagglutinin from A/VN/1203/04 (MVA-HA) (
16
) (
Fig. 2A
).
Additionally,
in vitro
functional analysis of H5M resulted in
hem-agglutination of RBCs at levels similar to those for wt HA (A.
Kamlangdee, B. Kingstad-Bakke, and J. E. Osorio, unpublished
data). These data thus demonstrate the successful generation of an
MVA vector expressing a mosaic H5N1 HA gene.
Efficacy of MVA-H5M vaccine against influenza viruses.
To
determine whether antibodies to MVA-H5M had
virus-neutraliz-ing activity, mice were intradermally inoculated with MVA-H5M,
MVA-LUC, or PBS; and the titers of antibodies against A/VN/
1203/04, A/MG/244/05, A/HK/483/97, and A/Egypt/1/08
chal-lenge viruses in immunized mice were measured. At 4 weeks
postimmunization, MVA-H5M elicited significant neutralizing
antibody (Ab) titers and protected against all four H5N1 strains
after challenge (
Fig. 2B
and
3A
to
C
). The geometric mean titers
(GMTs) of Abs against A/VN/1203/04, A/MG/244/05, A/HK/483/
97, and A/Egypt/1/08 did not differ significantly (
n
⫽
8 per group,
P
⬎
0.05) (
Fig. 2B
). None of animals injected with the MVA-LUC
control survived the challenge (
Fig. 3A
to
C
). Notably, no virus
replication was detected in the lungs of any of the
MVA-H5M-vaccinated mice challenged with any of the four H5N1 viruses
(
Fig. 3B
). Furthermore, vaccination with MVA-H5M reduced the
lung pathology after challenge with avian influenza viruses (
Fig.
4A
to
D
). MVA-H5M-vaccinated mice showed no to mild lung
lesions, whereas the MVA-LUC-vaccinated groups did show lung
lesions (
Fig. 4E
to
H
). Lesions included thickening of the alveolar
wall, lung consolidation with white blood cell infiltration, necrosis
of alveolar walls, and pulmonary edema. Immunohistochemistry
staining revealed large quantities of viral antigen in the MVA-LUC
control group (
Fig. 5E
to
H
). In contrast, viral antigen was not
detected in the lungs of mice that received MVA-H5M (
Fig. 5A
to
D
). These data demonstrate the ability of MVA-H5M to confer
both broad and strong protection against multiple clades of avian
influenza viruses.
We also evaluated the ability of the MVA-H5M construct to
protect mice against seasonal influenza viruses. Vaccination
fol-lowed the same protocol used with H5N1 viruses, except that mice
were challenged with A/Puerto Rico/8/1934 (PR8; H1N1) and
A/Aichi/2/1968 (H3N2). At 4 weeks postvaccination, the titers of
neutralizing Abs against both seasonal influenza viruses (
n
⫽
8 per
group) were below detectable levels (
Fig. 2B
). Despite the lack of
detectable neutralizing Ab, MVA-H5M conferred complete
pro-tection against PR8 (H1N1), with no significant weight loss being
observed (
Fig. 6A
and
C
). In contrast, no protection against
A/Ai-chi/2/68 (H3N2) challenge was observed (
Fig. 6A
and
C
).
Regard-less of the strain tested, viral replication was observed in the lungs
of mice vaccinated with MVA-H5M and challenged with seasonal
influenza viruses; however, mice challenged with PR8 had
signif-icantly lower viral lung titers than mice vaccinated with the
MVA-LUC control (
Fig. 6B
). In contrast, vaccination with MVA-H5M
FIG 4MVA-H5M reduces lung pathology after challenge with avian influenza viruses. Tissue sections of the lungs of mice vaccinated with MVA-H5M (A to D)
or MVA-LUC (E to H) and challenged with A/VN/1203/04 (A and E), A/MG/244/05 (B and F), A/HK/483/97 (C and G), or A/Egypt/1/08 (D and H) virus are shown. MVA-H5M-vaccinated mice showed normal to mild lung lesions, whereas MVA-LUC-vaccinated mice showed severe lung lesions, including lung consolidation, leukocyte infiltration, thickening of the alveolar septa, and alveolar edema.
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[image:5.585.79.513.66.220.2]had no effect on viral replication in the lungs of mice challenged
with A/Aichi/2/68 (
Fig. 6B
).
Short- and long-term immunity.
To assess the ability of the
MVA-H5M construct to confer both short- and long-term
immu-nities, groups of mice were vaccinated with a single dose of
MVA-H5M and then challenged at either 10 days or 6 months
postvac-cination. MVA-H5M provided full protection against a lethal dose
of A/HK/483/97 at both tested time points (
Fig. 7A
and
B
).
Neu-FIG 5MVA-H5M prevents viral replication in the lung after challenge with avian influenza viruses. Tissue sections of the lungs of mice vaccinated with
MVA-H5M (A to D) or MVA-LUC (E to H) and challenged with A/VN/1203/04 (A and E), A/MG/244/05 (B and F), A/HK/483/97 (C and G), or A/Egypt/1/08 (D and H) virus are shown. Lungs from mice were processed by immunohistochemistry with H5N1-specific antibody. Brown staining for viral antigen is indicated with arrowheads.
FIG 6MVA-H5M provides heterosubtypic protection against H1N1 virus. (A) Efficacies of a single dose of MVA-H5M or MVA-LUC vaccine against seasonal
H1N1 and H3N2 influenza viruses. MVA-H5M provided 100% protection with low weight loss against H1N1 virus but failed to prevent H3N2 virus infection. (B) MVA-H5M significantly lowered H1N1 viral replication in the lung compared to that in groups of mice that were challenged with H3N2 virus. (C) MVA-H5M prevented infection of vaccinated mice upon challenge with H1N1 virus, and the mice showed no to mild clinical signs and slight weight loss, but the vaccine failed to prevent infection upon challenge with H3N2 virus. Vaccinated mice were challenged at 5 weeks postvaccination, 2 mice per group were sacrificed at day 3 postinfection, and survival data were monitored for 14 days (n⫽6 mice per group).
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[image:6.585.79.509.66.217.2] [image:6.585.51.544.344.669.2]tralizing antibodies against A/HK/183/97 were also detected at
both 10 days and 6 months postvaccination (
Fig. 7C
).
Microneu-tralization assays and a challenge study were conducted using
A/HK/183/97 virus because it was the most virulent strain among
the four H5N1 strains used in the present study. Surprisingly, the
GMTs of Abs detected at 6 months postvaccination were
substan-tially higher than those detected at 4 weeks postvaccination (
Fig.
2B
and
7C
). In these animals, H5N1-specific IFN-
␥
-releasing
CD4
⫹and CD8
⫹T cell responses were detected using flow
cytom-etry. IFN-
␥
-releasing CD4
⫹and CD8
⫹T cells were found in
MVA-H5M-vaccinated mice 5 months after dosing (2 weeks
be-fore challenge in the long-term protection study), indicating a
long-term memory response (
Fig. 7D
).
Immune responses of mosaic and natural sequence vaccines.
We compared the immune responses to the mosaic vaccine
(MVA-H5M) to those to the wild-type or natural sequence
vac-cine (MVA-HA). Flow cytometry analyses of cytokine-producing
cells demonstrated that MVA-H5M-vaccinated mice had broader
and higher IFN-
␥
-producing CD4
⫹responses as well as broader
and higher IL-2-producing CD8
⫹T cell responses than mice
vac-cinated with MVA-HA (
Fig. 8A
and
B
) after stimulation with
di-verse H5 HA peptide pools. Furthermore, the T cell responses
against H1N1 (PR8) peptides were analyzed. The results showed
that MVA-H5M elicited CD8
⫹T cells responses to HA peptides
from H1N1 (PR8) virus, and the response was higher than that to
the MVA-HA wild-type vaccine (
Fig. 8C
). While MVA-H5M
elic-ited neutralizing antibodies against all viruses tested, MVA-HA
failed to elicit detectable responses against A/Egypt/1/08 virus
(
Fig. 8D
).
DISCUSSION
The rapid evolution of influenza viruses poses global health
chal-lenges necessitating the development of vaccines with broad
cross-protective immunity. Herein, we describe the development of a
broadly protective vaccine, MVA-H5M, based on a mosaic
epitope approach. The mosaic epitope approach minimizes
ge-netic differences between selected vaccine antigenic sequences and
circulating influenza virus strains, while it maximizes the overall
breadth of cross-protective immune responses. Our results
dem-onstrated that a single dose of MVA expressing a mosaic H5
hem-agglutinin (MVA-H5M) provided broad protection against
mul-tiple H5N1 viruses, including the highly pathogenic Egyptian
strains, and also an H1 subtype virus (PR8). The MVA-H5M
vac-cine provided robust and prolonged protection against a lethal
dose of a highly pathogenic avian influenza virus at as early as 10
days and as long as 6 months postvaccination.
In the past few years, commercially available vaccines have
failed to induce the expected level of protection against the
cur-rently circulating clade 2.2.1 in Egypt (
31
,
32
). It is very important
that an H5N1 influenza virus vaccine provide broad cross-clade
protection against these clade 2.2.1 viruses, particularly the
A/Egypt/1/08 strain, because this strain possess one of the four
mutations that are necessary to sustain human-to-human
trans-mission (
33
). The MVA-H5M vaccine provided complete
protec-tion against this H5N1 strain in mice. The ability to provide
com-plete protection against H5N1 viruses with a single dose is also
important for implementation; societal acceptance of a
single-dose vaccine would likely be higher than that of a multisingle-dose
vac-cine, especially during a pandemic.
On the basis of our data, there are several plausible hypotheses
to explain how our mosaic vaccine confers broad protection
against influenza virus challenge. One possible explanation is that
the 12-mer mosaic sequence captures more T helper cell epitopes,
in which case the broad protective ability of MVA-H5M likely
results from greater epitope coverage for the mosaic approach
than for previous approaches (
23
). This could translate to a higher
level of CD4
⫹T cells and antibody responses broader than those
induced by the wild-type sequence (MVA-HA). This hypothesis is
supported by the fact that MVA-H5M showed broader IFN-
␥
-FIG 7MVA-H5M provides short- and long-term immunities. (A and B) BALB/c mice were immunized with a single dose of MVA-H5M or MVA-LUC (n⫽7
mice per group). At 10 days or 6 months postvaccination, mice were challenged with a lethal dose of influenza virus A/HK/483/97. Survival data (A) and percent weight loss (B) were monitored for 14 days. (C) Neutralization titers from vaccinated mice at 10 days and 6 months postvaccination (n⫽7 mice per group). (D) CD4⫹and CD8⫹T cells responses at 5 months postvaccination.
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[image:7.585.103.485.68.283.2]producing CD4
⫹T cell epitope coverage and elicited broader
cross-clade neutralizing antibody responses (
Fig. 8A
,
B
, and
D
).
This MVA-H5M mosaic vaccine also elicited broader and higher
CD8
⫹T cell responses than the wild-type (MVA-HA) vaccine
(
Fig. 8B
). However, other immunological aspects of the
MVA-H5M vaccine still need to be further characterized. For example,
cytokine profiles, antibody epitope coverage, and mapping would
all be necessary to fully understand the mechanism responsible for
protection. Also, challenge studies comparing the mosaic
(MVA-H5M) vaccine and the wild-type vaccine or a natural sequence
(MVA-HA) vaccine will be conducted in the future. However, the
in vitro
data from T cell and neutralizing antibody responses
strongly indicate the broader immunogenicity of the mosaic H5
than the wild-type H5; i.e., the mosaic H5 elicited broader epitope
responses for both CD4
⫹and CD8
⫹against H5N1 peptides (
Fig.
8A
and
B
). It also elicited higher CD8
⫹T cell responses against
PR8 peptides (
Fig. 8C
) than wild-type H5 (MVA-HA).
Further-more, the mosaic H5 elicited broad neutralizing antibodies
against all 4 clades of avian influenza viruses that we tested,
in-cluding clade 0, clade 1, clade 2.2, and clade 2.2.1, whereas the
wild-type vaccine failed to elicit neutralizing antibodies against
clade 2.2.1 influenza virus A/Egypt/1/08, which is considered a
mutant strain (
31
,
32
,
34
). Commercially available vaccines also
failed to protect against this mutant Egyptian strain in Egypt in
recent years (
32
).
A second mechanism that may explain the breadth of
protec-tion conferred by the MVA-H5M vaccine is that the mosaic
ap-proach maintains an intact antigenic structure and, presumably,
physiological function (
35
). It has previously been reported that
most universal neutralizing antibodies are elicited by peptides in
the stalk regions (
36
,
37
). Our MVA-H5M vaccine has a normal
hemagglutination function and is also expressed as a cleavable
protein. Furthermore, our mosaic H5 might provide a higher level
of accessibility to the stalk region and stimulate a more robust
neutralizing antibody response against epitopes in the stalk
re-gion. Crystallography of the expressed mosaic H5 would likely be
required to reveal the actual structure of this protein and compare
it to the known structure of H5 hemagglutinin.
The MVA-H5M construct provided sterilizing lung protection
against H5N1 viruses, and no mortality or morbidity was detected
EGYPT /01 /08
FIG 8MVA-H5M elicits broader T cell epitope coverage, higher T cell responses, and broader neutralizing antibody responses than the wild-type
hemagglu-tinin-based vaccine. (A and B) Percentages of IFN-␥-producing CD4⫹T cells (A) and IL-2-producing CD8⫹T cells (B) from mice that were vaccinated with MVA-H5M or MVA-HA (n⫽3 mice per group). Splenocytes from vaccinated mice were collected and stimulated with HA peptide pools (indicated by p1 to p10) from H5N1 viruses. (C) IFN-␥-producing CD8⫹T cells from mice that were vaccinated with MVA-H5M or MVA-HA (n⫽3 mice per group). Splenocytes from vaccinated mice were collected and stimulated with HA peptide pools (indicated by p1 to p9) from H1N1 (PR8) virus. (D) Titers of neutralizing antibodies against H5N1 viruses elicited by the MVA-H5M and MVA-HA vaccines (n⫽7 mice per group). *,P⬍0.05, Student’sttest; **,P⬍0.01, Student’sttest.
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[image:8.585.71.519.64.423.2]play a role.
It is currently unclear whether the use of a live viral vector such
as MVA contributed to the increased cross protection described
herein. It is possible that H5M expression by MVA induced high
levels of cross-reactive neutralizing antibodies as well as CD4
⫹and CD8
⫹T cells via innate immune activations (
39
). In future
studies, we will evaluate the protective efficacy of the H5M antigen
as a recombinant protein without the MVA vector. This approach
will likely require the use of adjuvants and multiple doses to
achieve the desired protection.
The mosaic approach has previously been used for developing
vaccines against the highly variable HIV strains, capturing
poten-tial CD8
⫹T cell epitopes with a length of 9 amino acids (
23
,
26
)
while still maintaining a normal protein structure. Because
com-plete protection against influenza viruses is based primarily on
humoral immunity (
40
), we modified the algorithm for epitopes
of 12 amino acids in order to capture potential T helper cell
epitopes (
27
) in order to target antibody-producing plasma cell
via T helper cell activation. We speculate that this strategy may
have facilitated achievement of a high neutralizing antibody titer
with a single dose of MVA-H5M (
Fig. 2B
).
The vaccine elicited strong humoral responses against multiple
H5N1 viruses but no cross-neutralizing antibodies against
sea-sonal influenza viruses (H1N1 and H3N2). Despite the lack of
neutralizing antibodies against H1N1 PR8 virus, the MVA-H5M
vaccine provided 100% protection against the PR8 virus. This
sug-gests a substantial role of cellular immune responses against the
PR8 virus, as shown in
Fig. 8C
, likely because the H5M protein
possesses some cytotoxic T lymphocyte epitopes of the PR8 virus
(
35
). This protection can also be explained by the genetic
relation-ship between the H5 and H1 hemagglutinin subtypes, as both
belong to group 1 influenza A virus hemagglutinin (
41
), and it
elicits a large amount of nonneutralizing antibody which then
targets and destroys H1N1 virus via an antibody-dependent
cel-lular cytotoxicity (ADCC) mechanism (
42
). However, the
MVA-H5M vaccine did not protect immunized mice against influenza
virus A/Aichi/2/68, which is likely due to antigenic differences, as
H3 belongs to group 2 influenza A virus hemagglutinin. Because
the MVA vector can be designed to contain multiple inserts,
fu-ture constructs will contain mosaics from several hemagglutinin
subtypes, including important seasonal (e.g., H3) and emerging
(e.g., H7) pathogens. Since this vaccine provides broad protection
and a long duration of immunity, utilizing an MVA vector
ex-pressing seasonal mosaics might diminish the need for annual
vaccination.
The ability of the MVA-H5M vaccine to confer broad
protec-tive immunity against various heterologous strains as well as
het-erosubtypic strains makes the mosaic approach a very promising
strategy to combat the antigenic diversity of influenza viruses.
Taken together with codon optimization of HA for a high level of
protein expression and the use of an MVA vector as a backbone for
cellular immunity activation, this approach promises to increase
assistance with T cell analyses and Matthew Aliota (Pathobiological Sci-ences, University of Wisconsin—Madison, Madison, WI, USA) for criti-cal review of the manuscript.
A.K., B.K.-B., and J.E.O. conceived and designed the experiments. A.K. and B.K.-B. performed the experiments. A.K. analyzed the data. T.K.A., T.L.G., and J.E.O. contributed reagents, materials, and analysis tools. A.K., B.K.-B., and J.E.O. wrote the paper.
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